CN107796681B - 一种真菌荧光染色液及其制备方法 - Google Patents
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Abstract
本发明公开了一种真菌荧光染色液，由以下重量份(％)的原料组成：5‑羧基荧光素0.008‑0.012；真菌引物ITS40.045‑0.055；氢氧化钾8‑13；甘油28‑33；双磷脂酰甘油0.45‑0.55；抗坏血酸0.07‑0.13；水53.363‑63.427；本发明通过加入5‑羧基荧光素和真菌引物ITS4，在游离的状态下真菌引物ITS4形成茎环结构可有效避免荧光衰退，在氢氧化钾溶液中5‑羧基荧光素可穿透真菌细胞壁及细胞膜与真菌核糖体核糖核酸基因间隔区碱基发生特异性的结合，此时茎环结构打开在荧光显微镜下镜检呈现荧光；氢氧化钾在染色过程中熔化真菌角质层使真菌引物ITS4快速进入真菌细胞内作用；另外，由于配方中加入了抗荧光衰退剂，使荧光引物与真菌核糖体核糖核酸基因间隔区碱基特异性结合后能长期保持。
Description
技术领域
本发明涉及医学临床检验中病原菌生物学检验技术领域，具体涉及一种用于真菌菌体染色的真菌荧光染色液及其制备方法。
背景技术
现在医学临床检验中，对真菌感染检验大多采用传统的氢氧化钾法或近几年发展的免疫学试验法。其中：
氢氧化钾法虽操作较为简便，但存在诸多不足之处，其一是不具有特异性，很容易受到其他杂质底物的影响，导致观察不清晰易出现假阴性现象；其二是检验过程中必须找到真菌菌丝或孢子结构才能得到检验结论，因此需消耗大量检验时间，工作效率低下；其三是该方法检验范围有限，对糠秕孢子菌、马拉色菌根本不起作用。
免疫学试验法虽具有很好的特异性，但是只可针对深部真菌感染的抗体进行检验，其缺点在于：其一、对因免疫系统功能降低不出现抗体的系统性感染者不能够及时检出；其二、对已感染康复过程中产生抗体维持时间较长的患者，这类人群中有一定的假阳性率出现。因此，该方法未能得到广泛应用。
随着医疗检验水平的不断推进，近年来真菌荧光染色临床检验技术逐渐推广，目前市场上大多采用细胞壁β-多糖、几丁质等荧光染色法，该方法只通过荧光染料与细胞壁β-多糖、几丁质等以亲和力结合，不具有特异性；随着临床逐渐推广发现，易受到染色底物杂质的干扰，影响观察结果的判断；另外，荧光衰退较快，样本无法保存；这给临床推广及应用造成非常大的困扰。
基于以上背景下，本发明较好的解决了上述难题，这正是本发明的价值所在。
发明内容
针对医学临床检验中病原菌生物学检验，为能够快速准确的检验致病真菌，本发明提供一种真菌荧光染色液以5-羧基荧光素与真菌引物I TS4结合形成荧光引物，与真菌核糖体核糖核酸基因间隔(ITS)区碱基发生特异性的结合将真菌标记荧光，用荧光显微镜在480nm波长下镜检观察背景为黑色，真菌发射出520nm波长的绿色荧光。
本发明一种真菌荧光染色液，由以下重量份(％)的原料组成：
优选地，本发明一种真菌荧光染色液，由以下重量份(％)的原料组成：
一种真菌荧光染色液的制备方法，包括以下步骤：
1)用电子天平准确称取5-羧基荧光素、真菌引物ITS4、水于500ml三角烧瓶中，用玻璃棒轻轻搅拌充分溶解后，再称取氢氧化钾倒入烧瓶中，轻轻搅拌充分溶解后盖上塞子；
2)将上述溶液放置于37℃恒温摇床，缓慢振摇1小时取出，此为溶液1；
3)用电子天平准确称取甘油、双磷脂酰甘油、抗坏血酸、水于1L三角烧瓶中，用玻璃棒搅拌充分溶解，此为溶液2；
4)将溶液1缓慢倒入溶液2中，边倒入边搅拌使其充分混合后，即得本发明真菌荧光染色液，置于棕色瓶中保存。
本发明是这样实现的，通过将上述原料按重量份配制成荧光染色液，荧光染色液中5-羧基荧光素的羧基与真菌核糖体核糖核酸间隔区引物4(ITS4)的核苷酸链两端的磷酸基团端氨基残基共价结合标记成荧光引物，在游离状态时该荧光引物形成茎环结构避免5-羧基荧光素衰退，在氢氧化钾溶液中快速进入真菌细胞与真菌核糖体核糖核酸基因间隔区(ITS)碱基发生特异性的结合，此时，茎环结构打开，露出5-羧基荧光素将真菌细胞标记荧光，另外，甘油、双磷脂酰甘油、抗坏血酸形成的抗荧光衰退保护层将露出5-羧基荧光素封闭式包裹阻止荧光衰退，使真菌样本能长期保存，用荧光显微镜在480nm波长下镜检观察背景为黑色，真菌发射出520nm波长的绿色荧光。
与现有技术相比，本发明的有益效果是：
本发明通过加入5-羧基荧光素和真菌引物ITS4，在游离的状态下真菌引物ITS4形成茎环结构可有效避免荧光衰退，在氢氧化钾溶液中5-羧基荧光素可穿透真菌细胞壁及细胞膜与真菌核糖体核糖核酸(rRNA)基因间隔(ITS)区碱基发生特异性的结合，此时茎环结构打开在荧光显微镜下镜检呈现荧光；氢氧化钾在染色过程中熔化真菌角质层使真菌引物ITS4快速进入真菌细胞内作用；另外，由于配方中加入了抗荧光衰退剂(甘油、双磷脂酰甘油、抗坏血酸)，使荧光引物与真菌核糖体核糖核酸(rRNA)基因间隔(ITS)区碱基特异性结合后能长期保持，同时也增加了目标真菌在镜检时发出的荧光强度，使目标真菌标本与背景之间的对比度大大增强，在镜检过程中使目标真菌更加显目。因此，本发明的优点在于使用操作简便、观察清晰度高、特异性好、结果准确，有较好的临床应用前景；在临床检验过程中操作简便、效率高、使用范围全面、特异性好、准确度高；
检验灵敏度高，减少检验结果假阴性的发生，进而降低漏检率；视野清晰，反应为荧光反应，在检验视野中以清晰的荧光呈现，便于观察查找，大大提高工作效率；抗干扰能力强，反应为特异性结合，杂质和非真菌细胞不会被荧光标记，消除了杂质和非真菌细胞对检验过程中的干扰，使目标更加显目易观察；荧光保持时间长，加入了抗荧光衰减剂形成保护层对5-羧基荧光素进行封闭式包裹，使荧光染料不与外界接触，从而很好的保护了荧光染料，使荧光持久不衰减，使标本可以保存至相关规定保存期限。
附图说明
图1为实施例1制得的真菌荧光染色液染色结果图。
图2为KOH湿片法染色结果图。
图3为市场现有的真菌荧光染色液染色结果图。
图4为实施例1制得的真菌荧光染色液在制片当天染色结果图。
图5为实施例1制得的真菌荧光染色液在12个月后染色结果图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例1
本发明一种真菌荧光染色液，由以下重量份(％)的原料组成：
真菌引物ITS4的全称为真菌核糖体核糖核酸间隔区引物4，这里简称真菌引物ITS4。
5-羧基荧光素在这里简称5-FAM。
一种真菌荧光染色液的制备方法，包括以下步骤：
1)用万分之一电子天平准确称取5-羧基荧光素0.1g、真菌引物I TS40.5g、水250g于500ml具赛三角烧瓶中，用玻璃棒轻轻搅拌充分溶解后，再称取氢氧化钾100g倒入烧瓶中，轻轻搅拌充分溶解后盖上塞子；
2)将上述溶液放置于37℃恒温摇床，缓慢振摇1小时取出，此为溶液1；
3)用万分之一电子天平准确称取甘油300g、双磷脂酰甘油5g、抗坏血酸1g、水343.4g于1L具赛三角烧瓶中，用玻璃棒搅拌充分溶解，此为溶液2；
4)将溶液1缓慢倒入溶液2中，边到边搅拌使其充分混合后，即得本发明真菌染色液，置于棕色瓶中保存。
本实施例1真菌荧光染色液的使用方法：
取头皮屑样本于载玻片上，直接向样本滴加一滴染色液(以染色液覆盖或淹没样本为准)，盖上盖玻片，使用棉棒或纸巾压片，吸去多余染色液，将载玻片放在荧光显微镜载物台上，在480nm波长下观察，观察时视野背景为黑色，真菌放射出520nm波长的绿色荧光亮点。
本发明是这样实现的，通过将上述原料按重量份配制成荧光染色液，荧光染色液中5-羧基荧光素的羧基与真菌引物ITS4的5’末端氨基残基共价结合标记成荧光引物，在游离状态时该荧光引物形成茎环结构避免5-羧基荧光素衰退，在氢氧化钾溶液中快速进入真菌细胞与真菌核糖体核糖核酸基因间隔区(ITS)碱基发生特异性的结合，此时，茎环结构打开，露出5-羧基荧光素将真菌细胞标记荧光，另外，甘油、双磷脂酰甘油、抗坏血酸形成的抗荧光衰退保护层将露出5-羧基荧光素封闭式包裹阻止荧光衰退，使真菌样本能长期保存，用荧光显微镜在480nm波长下镜检观察背景为黑色，真菌发射出520nm波长的绿色荧光。
实施例2
相关性实验：
(1)取头皮屑样本用KOH湿片法进行检测，具体步骤为：取头皮屑置载玻片上，滴加10％KOH溶液，盖上盖玻片，在酒精灯火焰上稍加热，待标本溶解，轻轻加压盖玻片使标本透明，将载玻片放在显微镜载物台上，先用低倍镜找到视野，再切换至高倍镜观察。结果见附图2；
(2)采用合肥华今生物科技有限公司生产的一批批号为2016061301的真菌荧光染色液进行荧光染色检测，结果见附图3；
(3)采用实施例1所得荧光染色液进行检验，具体操作方法与实施例1使用方法相同，结果见附图1。
从附图2可以看出用氢氧化钾法对头皮屑检测时无法观察到真菌菌丝或真菌孢子，结果为阴性；从附图2可以看出，市场现有一种真菌荧光染色法虽能看到真菌，但受背景影响，真菌形态不理想，辨认度不高，容易判断错误；从附图1可以看出，实施例1所得荧光染色液通过荧光显微镜可以清晰看到真菌形态及大小，观察时一目了然。
实施例3
5-羧基荧光素荧光持久性实验：取足癣患者患处皮屑样本用实施例1所得荧光染色液进行实验，结果见附图4和5。
从附图4和5可以看出实施例1所得荧光染色液同一制片在制片当天和12个月后观察结果没有差别。
这个实验说明抗荧光衰退剂能够形成保护层对5-羧基荧光素进行封闭式包裹，使荧光染料不与外界的荧光淬灭物质(重金属离子、卤素离子、溶解氧)接触，很好的保护了荧光染料，使本发明制得的真菌荧光染色液荧光持久不衰退。
Claims (3)
1.一种真菌荧光染色液，其特征在于，由以下重量份(％)的原料组成：
2.根据权利要求1所述的真菌荧光染色液，其特征在于，由以下重量份(％)的原料组成：
3.如权利要求1或2所述的真菌荧光染色液的制备方法，其特征在于，包括以下步骤：
1)用电子天平准确称取5-羧基荧光素、真菌引物ITS4、水于500ml三角烧瓶中，用玻璃棒轻轻搅拌充分溶解后，再称取氢氧化钾倒入烧瓶中，轻轻搅拌充分溶解后盖上塞子；
2)将上述溶液放置于37℃恒温摇床，缓慢振摇1小时取出，此为溶液1；
3)用电子天平准确称取甘油、双磷脂酰甘油、抗坏血酸、水于1L三角烧瓶中，用玻璃棒搅拌充分溶解，此为溶液2；
4)将溶液1缓慢倒入溶液2中，边倒入边搅拌使其充分混合后，即得真菌荧光染色液，置于棕色瓶中保存。
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